Ophthalmic compositions for treating ocular hypertension

ABSTRACT

This invention relates to potent potassium channel blocker compounds of Formula I or a formulation thereof for the treatment of glaucoma and other conditions which leads to elevated intraocular pressure in the eye of a patient. This invention also relates to the use of such compounds to provide a neuroprotective effect to the eye of mammalian species, particularly humans.

This claims the benefit of U.S. Provisional Application 60/424,808 and60/500,091.

BACKGROUND OF THE INVENTION

Glaucoma is a degenerative disease of the eye wherein the intraocularpressure is too high to permit normal eye function. As a result, damagemay occur to the optic nerve head and result in irreversible loss ofvisual function. If untreated, glaucoma may eventually lead toblindness. Ocular hypertension, i.e., the condition of elevatedintraocular pressure without optic nerve head damage or characteristicglaucomatous visual field defects, is now believed by the majority ofophthalmologists to represent merely the earliest phase in the onset ofglaucoma.

There are several therapies for treating glaucoma and elevatedintraocular pressure, but the efficacy and the side effect profiles ofthese agents are not ideal. Recently potassium channel blockers werefound to reduce intraocular pressure in the eye and therefore provideyet one more approach to the treatment of ocular hypertension and thedegenerative ocular conditions related thereto. Blockage of potassiumchannels can diminish fluid secretion, and under some circumstances,increase smooth muscle contraction and would be expected to lower IOPand have neuroprotective effects in the eye. (see U.S. Pat. Nos.5,573,758 and 5,925,342; Moore, et al., Invest. Ophthalmol. Vis. Sci 38,1997; WO 89/10757, WO94/28'900, and WO 96/33719).

SUMMARY OF THE INVENTION

This invention relates to the use of potent potassium channel blockersor a formulation thereof in the treatment of glaucoma and otherconditions which are related to elevated intraocular pressure in the eyeof a patient. This invention also relates to the use of such compoundsto provide a neuroprotective effect to the eye of mammalian species,particularly humans. More particularly this invention relates to thetreatment of glaucoma and/or ocular hypertension (elevated intraocularpressure) using novel indazole compounds having the structural formulaI:

or a pharmaceutically acceptable salt, enantiomer, diastereomer ormixture thereof:wherein,R represents hydrogen, or C₁₋₆ alkyl;X represents —(CHR₇)_(p)—, —(CHR₇)_(p)CO—;Y represents —CO(CH₂)_(n)—, CH₂, or —CH(OR)—;Q represents CR^(y);R^(y) represents H, or C₁₋₆ alkyl;R_(w) represents H, C₁₋₆ alkyl, —C(O)C₁₋₆ alkyl, —C(O)OC₁₋₆ alkyl,—SO₂N(R)₂, —SO₂C₁₋₆ alkyl, —SO₂C₆₋₁₀ aryl, NO₂, CN or —C(O)N(R)₂;R₂ represents hydrogen, C₁₋₁₀ alkyl, OH, C₂₋₆ alkenyl, C₁₋₆ alkylSR,—(CH₂)_(n)O(CH₂)_(m)OR, —(CH₂)_(n)C₁₋₆ alkoxy, —(CH₂)_(n)C₃₋₈cycloalkyl, —(CH₂)_(n)C₃₋₁₀ heterocyclyl, —N(R)₂, —COOR, or—(CH₂)_(n)C₆₋₁₀ aryl, said alkyl, heterocyclyl, or aryl optionallysubstituted with 1-3 groups selected from R^(a);R₃ represents hydrogen, C₁₋₁₀ alkyl, —(CH₂)_(n)C₃₋₈ cycloalkyl,—(CH₂)_(n)C₃₋₁₀ heterocyclyl, —(CH₂)_(n)COOR, —(CH₂)_(n)C₆₋₁₀ aryl,—(CH₂)_(n)NHR₈, —(CH₂)_(n)N(R)₂, —(CH₂)_(n)N(R₈)₂, —(CH₂)_(n)NHCOOR,—(CH₂)_(n)N(R₈)CO₂R, —(CH₂)_(n)N(R₈)COR, —(CH₂)_(n)NHCOR₂,—(CH₂)_(n)CON(R₈), aryl, —(CH₂)_(n)C₁₋₆ alkoxy, CF₃, —(CH₂)_(n)SO₂R,—(CH₂)_(n)SO₂N(R)₂, —(CH₂)_(n)CON(R)₂, —(CH₂)_(n)CONHC(R)₃,—(CH₂)_(n)CONHC(R)₂CO₂R, —(CH₂)_(n)COR₈, nitro, cyano or halogen, saidalkyl, alkoxy, heterocyclyl, or aryl optionally substituted with 1-3groups of R^(a);or R₂ and R₃ taken together with the intervening Q form a 3-10 memberedcarbocyclic or heterocyclic carbon ring optionally interrupted by 1-2atoms of O, S, C(O) or NR, and optionally having 1-4 double bonds, andoptionally substituted by 1-3 groups selected from R^(a);or R₂ and R₃ taken together with the intervening Q represent OR;R₄ and R₅ independently represent hydrogen, C₁₋₆ alkoxy, OH, C₁₋₆ alkyl,COOR, SO_(q)C₁₋₆ alkyl, COC₁₋₆ alkyl, SO₃H, —O(CH₂)_(n)N(R)₂,—O(CH₂)_(n)CO₂R, —OPO(OH)₂, CF₃, OCF₃—N(R)₂, nitro, cyano, C₁₋₆alkylamino, or halogen; andR₆ represents hydrogen, C₁₋₁₀ alkyl, —(CH₂)_(n)C₆₋₁₀ aryl, NR_(c)R_(d),—NR(CH₂)_(n)C₆₋₁₀ aryl, —N((CH₂)_(n)C₆₋₁₀ aryl)₂, —(CH₂)_(n)C₃₋₁₀heterocyclyl, —NR(CH₂)_(n)C₃₋₁₀ heterocyclyl, —N((CH₂)_(n)C₃₋₁₀heterocyclyl)₂ (C₆₋₁₀ aryl)O—, —(CH₂)_(n)C₃₋₈ cycloalkyl, —COOR,—C(O)CO₂R, said aryl, heterocyclyl and alkyl optionally substituted with1-3 groups selected from R^(a), wherein the R^(a)(s) can be attached toany carbon atom or heteroatom selected from N and S;R_(c) and R_(d) independently represent H, C₁₋₆ alkyl, C₂₋₆ alkenyl,C₁₋₆ alkylSR, —(CH₂)_(n)O(CH₂)_(m)OR, —(CH₂)_(n)C₁₋₆ alkoxy, or—(CH₂)_(n)C₃₋₈ cycloalkyl;or R_(c) and R_(d) taken together with the intervening N atom form a4-10 membered heterocyclic carbon ring optionally interrupted by 1-2atoms of O, S, C(O) or NR, and optionally having 1-4 double bonds, andoptionally substituted by 1-3 groups selected from R^(a);R₇ represents hydrogen, C₁₋₆ alkyl, —(CH₂)_(n)COOR or —(CH₂)_(n)N(R)₂,R₈ represents —(CH₂)_(n)C₃₋₈ cycloalkyl, —(CH₂)_(n)3-10 heterocyclyl,C₁₋₆ alkoxy or —(CH₂)_(n)C₅₋₁₀ heteroaryl, —(CH₂)_(n)C₆₋₁₀ aryl saidheterocyclyl, aryl or heteroaryl optionally substituted with 1-3 groupsselected from R^(a);R^(a) represents F, Cl, Br, I, CF₃, N(R)₂, NO₂, CN, —O—, —COR₈, —CONHR₈,—CON(R₈)₂, —O(CH₂)_(n)COOR, —NH(CH₂)_(n)OR, —COOR, —OCF₃, CF₂CH₂OR,—NHCOR, —SO₂R, —SO₂NR₂, —SR, (C₁-C₆ alkyl)O—, —(CH₂)_(n)O(CH₂)_(m)OR,—(CH₂)_(n)C₁₋₆ alkoxy, (aryl)O—, —(CH₂)_(n)OH, (C₁-C₆ alkyl)S(O)_(m)—,H₂N—C(NH)—, (C₁-C₆ alkyl)C(O)—, (C₁-C₆ alkyl)OC(O)NH—, —(C₁-C₆alkyl)NR_(w)(CH₂)_(n)C₃₋₁₀ heterocyclyl-R_(w), —(C₁-C₆alkyl)O(CH₂)_(n)C₃₋₁₀ heterocyclyl-R_(w), —(C₁-C₆ alkyl)S(CH₂)_(n)C₃₋₁₀heterocyclyl-R_(w), —(C₁-C₆ alkyl)-C₃₋₁₀ heterocyclyl-R_(w),—(CH₂)_(n)—Z¹—C(═Z²)N(R)₂, —(C₂₋₆ alkenyl)NR_(w)(CH₂)_(n)C₃₋₁₀heterocyclyl-R_(w), —(C₂₋₆ alkenyl)O(CH₂)_(n)C₃₋₁₀ heterocyclyl-R_(w),—(C₂₋₆ alkenyl)S(CH₂)_(n)C₃₋₁₀ heterocyclyl-R_(w), —(C₂₋₆ alkenyl)-C₃₋₁₀heterocyclyl-R_(w), —(C₂₋₆ alkenyl)—Z¹—C(═Z²)N(R)₂, —(CH₂)_(n)SO₂R,—(CH₂)_(n)SO₃H, —(CH₂)_(n)PO(OR)₂, C₃₋₁₀cycloalkyl, C₆₋₁₀ aryl, C₃₋₁₀heterocyclyl, C₂₋₆ alkenyl, and C₁-C₁₀ alkyl, said alkyl, alkenyl,alkoxy, heterocyclyl and aryl optionally substituted with 1-3 groupsselected from C₁-C₆ alkyl, halogen, (CH₂)_(n)OH, CN, NO₂, CON(R)₂ andCOOR;Z¹ and Z² independently represents NR_(w), O, CH₂, or S;m is 0-3;n is 0-3;p is 0-3 andq is 0-2.

This and other aspects of the invention will be realized upon inspectionof the invention as a whole.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to novel potassium channel blockers ofFormula I. It also relates to a method for decreasing elevatedintraocular pressure or treating glaucoma by administration, preferablytopical or intra-camaral administration, of a composition containing apotassium channel blocker of Formula I described hereinabove and apharmaceutically acceptable carrier.

One embodiment of this invention is realized when p is 1-3.

Another embodiment of this invention is realized when Y is —CO(CH₂)_(n)and all other variables are as originally described. A subembodiment ofthis invention is realized when n is 0.

Another embodiment of this invention is realized when Y is CH(OR) andall other variables are as originally described.

In another embodiment R_(w) is selected from H, C₁₋₆ alkyl, —C(O)C₁₋₆alkyl and —C(O)N(R)₂ and all other variables are as originallydescribed.

In another embodiment X is —(CHR₇)_(p)—, p is 1-3 and all othervariables are as originally described.

In another embodiment X is —(CHR₇)_(p)CO—, p is 1-3 and all othervariables are as originally described.

Still another embodiment of this invention is realized when R₆ is C₁₋₁₀alkyl, (CH₂)_(n)C₆₋₁₀ aryl, (CH₂)_(n)C₃₋₁₀ heterocyclyl, NR_(c)R_(d) or(CH₂)_(n)C₃₋₈ cycloalkyl, said alkyl, aryl, heterocyclyl and cycloalkyloptionally substituted with 1 to 3 groups of R^(a), and all othervariables are as originally described.

Yet another embodiment of this invention is realized when R₆ is C₁₋₁₀alkyl, (CH₂)_(n)C₆₋₁₀ aryl, or (CH₂)_(n)C₃₋₁₀ heterocyclyl, said alkyl,aryl, and heterocyclyl optionally substituted with 1 to 3 groups ofR^(a), and all other variables are as originally described.

Yet another embodiment of this invention is realized when R₇ is hydrogenor C₁₋₆ alkyl, and all other variables are as originally described.

Yet another embodiment of this invention is realized when Y is—CO(CH₂)_(n), and n is 0.

Still another embodiment of this invention is realized when Y is—CO(CH₂)_(n), R₂ is C₁₋₁₀ alkyl or C₁₋₆ alkylOR and R₃ is C₁₋₁₀ alkyl,(CH₂)_(n)C₃₋₁₀ heterocyclyl, X is —(CHR₇)_(p)CO—, and p is 1-3 saidheterocyclyl and alkyl optionally substituted with 1 to 3 groups ofR^(a). A subembodiment of this invention is realized when n is 0.

Another embodiment of the instant invention is realized when R^(a) isselected from F, Cl, Br, I, CF₃, N(R)₂, NO₂, CN, —O—, —CONHR₈,—CON(R₈)₂, —O(CH₂)_(n)COOR, —NH(CH₂)_(n)OR, —COOR, —OCF₃, —NHCOR, —SO₂R,—SO₂NR₂, —SR, (C₁-C₆ alkyl)O—, —(CH₂)_(n)O(CH₂)_(m)OR, —(CH₂)_(n)C₁₋₆alkoxy, (aryl)O—, —OH, (C₁-C₆ alkyl)S(O)_(m)—, H₂N—C(NH)—, (C₁-C₆alkyl)C(O)—, (C₁-C₆ alkyl)OC(O)NH—, —(C₁-C₆ alkyl)NR_(w)(CH₂)_(n)C₃₋₁₀heterocyclyl-R_(w), —(CH₂)_(n)—Z¹—C(═Z²)N(R)₂, —(C₂₋₆alkenyl)NR_(w)(CH₂)_(n)C₃₋₁₀ heterocyclyl-R_(w), —(C₂₋₆alkenyl)—Z¹—C(═Z²)N(R)₂, —(CH₂)_(n)SO₂R, —(CH₂)_(n)SO₃H,—(CH₂)_(n)PO(OR)₂, C₂₋₆ alkenyl, and C₁-C₁₀ alkyl, said alkyl andalkenyl, optionally substituted with 1-3 groups selected fromC₁-C₆alkyl, and COOR;

Examples of compounds to be used in this invention are found in Tables 1and 2:

TABLE 1

R1 R2

TABLE 2

-   -   wherein R₁ is

and X is N or CH;or a pharmaceutically acceptable salt, enantiomer, diastereomer ormixture thereof.

The invention is described herein in detail using the terms definedbelow unless otherwise specified.

The compounds of the present invention may have asymmetric centers,chiral axes and chiral planes, and occur as racemates, racemic mixtures,and as individual diastereomers, with all possible isomers, includingoptical isomers, being included in the present invention. (See E. L.Eliel and S. H. Wilen Stereochemistry of Carbon Compounds (John Wileyand Sons, New York 1994), in particular pages 1119-1190)

When any variable (e.g. aryl, heterocycle, R¹, R⁶ etc.) occurs more thanone time in any constituent, its definition on each occurrence isindependent at every other occurrence. Also, combinations ofsubstituents/or variables are permissible only if such combinationsresult in stable compounds.

When R^(a) is —O— and attached to a carbon it is referred to as acarbonyl group and when it is attached to a nitrogen (e.g., nitrogenatom on a pyridyl group) or sulfur atom it is referred to a N-oxide andsulfoxide group, respectively.

The term “alkyl” refers to a monovalent alkane (hydrocarbon) derivedradical containing from 1 to 10 carbon atoms unless otherwise defined.It may be straight, branched or cyclic. Preferred alkyl groups includemethyl, ethyl, propyl, isopropyl, butyl, t-butyl, cyclopropylcyclopentyl and cyclohexyl. When the alkyl group is said to besubstituted with an alkyl group, this is used interchangeably with“branched alkyl group”.

Cycloalkyl is a specie of alkyl containing from 3 to 15 carbon atoms,unless otherwise defined, without alternating or resonating double bondsbetween carbon atoms. It may contain from 1 to 4 rings, which are fused.Examples of such cycloalkyl elements include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

Alkenyl is C₂-C₆ alkenyl.

Alkoxy refers to an alkyl group of indicated number of carbon atomsattached through an oxygen bridge, with the alkyl group optionallysubstituted as described herein. Said groups are those groups of thedesignated length in either a straight or branched configuration and iftwo or more carbon atoms in length, they may include a double or atriple bond. Exemplary of such alkoxy groups are methoxy, ethoxy,propoxy, isopropoxy, butoxy, isobutoxy, tertiary butoxy, pentoxy,isopentoxy, hexoxy, isohexoxy allyloxy, propargyloxy, and the like.

Halogen (halo) refers to chlorine, fluorine, iodine or bromine.

Aryl refers to aromatic rings e.g., phenyl, substituted phenyl and thelike, as well as rings which are fused, e.g., naphthyl, phenanthrenyland the like. An aryl group thus contains at least one ring having atleast 6 atoms, with up to five such rings being present, containing upto 22 atoms therein, with alternating (resonating) double bonds betweenadjacent carbon atoms or suitable heteroatoms. Examples of aryl groupsare phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl,phenanthryl, anthryl or acenaphthyl and phenanthrenyl, preferablyphenyl, naphthyl or phenanthrenyl. Aryl groups may likewise besubstituted as defined. Preferred substituted aryls include phenyl andnaphthyl.

The term heterocyclyl or heterocyclic, as used herein, represents astable 3- to 7-membered monocyclic or stable 8- to 11-membered bicyclicheterocyclic ring which is either saturated or unsaturated, and whichconsists of carbon atoms and from one to four heteroatoms selected fromthe group consisting of N, O, and S, and including any bicyclic group inwhich any of the above-defined heterocyclic rings is fused to a benzenering. The heterocyclic ring may be attached at any heteroatom or carbonatom which results in the creation of a stable structure. A fusedheterocyclic ring system may include carbocyclic rings and need includeonly one heterocyclic ring. The term heterocycle or heterocyclicincludes heteroaryl moieties. Examples of such heterocyclic elementsinclude, but are not limited to, azepinyl, benzimidazolyl,benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl,benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl,cinnolinyl, dihydrobenzofuryl, dihydrobenzothienyl,dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone,dihydropyrrolyl, 1,3-dioxolanyl, furyl, imidazolidinyl, imidazolinyl,imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl,isoquinolinyl, isothiazolidinyl, isothiazolyl, isothiazolidinyl,morpholinyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl,2-oxopiperazinyl, 2-oxopiperdinyl, 2-oxopyrrolidinyl, piperidyl,piperazinyl, pyridyl, pyrazinyl, pyrazolidinyl, pyrazolyl, pyridazinyl,pyrimidinyl, pyrrolidinyl, pyrrolyl, quinazolinyl, quinolinyl,quinoxalinyl, tetrahydrofuryl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, thiamorpholinyl, thiamorpholinyl sulfoxide,thiazolyl, thiazolinyl, thienofuryl, thienothienyl, and thienyl.Preferably, heterocycle is selected from 2-azepinonyl, benzimidazolyl,2-diazapinonyl, dihydroimidazolyl, dihydropyrrolyl imidazolyl,2-imidazolidinonyl, indolyl, isoquinolinyl, morpholinyl, piperidyl,piperazinyl, pyridyl, pyrrolidinyl, 2-piperidinonyl, 2-pyrimidinonyl,2-pyrollidinonyl, quinolinyl, tetrahydrofuryl, tetrahydroisoquinolinyl,and thienyl.

The term “heteroatom” means, O, S or N, selected on an independentbasis.

The term “heteroaryl” refers to a monocyclic aromatic hydrocarbon grouphaving 5 or 6 ring atoms, or a bicyclic aromatic group having 8 to 10atoms, containing at least one heteroatom, O, S or N, in which a carbonor nitrogen atom is the point of attachment, and in which one or twoadditional carbon atoms is optionally replaced by a heteroatom selectedfrom O or S, and in which from 1 to 3 additional carbon atoms areoptionally replaced by nitrogen heteroatoms, said heteroaryl group beingoptionally substituted as described herein. Examples of suchheterocyclic elements include, but are not limited to, benzimidazolyl,benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl,benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl,cinnolinyl, dihydrobenzofuryl, dihydrobenzothienyl,dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone, furyl,imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl,isoquinolinyl, isothiazolyl, naphthyridinyl, oxadiazolyl, pyridyl,pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl, pyrrolyl, quinazolinyl,quinolinyl, quinoxalinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,thiazolyl, thienofuryl, thienothienyl, thienyl and triazolyl. Additionalnitrogen atoms may be present together with the first nitrogen andoxygen or sulfur, giving, e.g., thiadiazole.

This invention is also concerned with compositions and methods oftreating ocular hypertension or glaucoma by administering to a patientin need thereof one of the compounds of formula I in combination with aβ-adrenergic blocking agent such as timolol, betaxolol, levobetaxolol,carteolol, levobunolol, a parasympathomimetic agent such as epinephrine,iopidine, brimonidine, clonidine, para-aminoclonidine, carbonicanhydrase inhibitor such as dorzolamide, acetazolamide, metazolamide orbrinzolamide, an EP4 agonist (such as those disclosed in WO 02/24647, WO02/42268, EP 1114816, WO 01/46140 and WO 01/72268), a prostaglandin suchas latanoprost, travaprost, unoprostone, rescula, S1033 (compounds setforth in U.S. Pat. Nos. 5,889,052; 5,296,504; 5,422,368; and 5,151,444);a hypotensive lipid such as lumigan and the compounds set forth in U.S.Pat. No. 5,352,708; a neuroprotectant disclosed in U.S. Pat. No.4,690,931, particularly eliprodil and R-eliprodil as set forth in WO94/13275, including memantine; or an agonist (if 5-HT2 receptors as setforth in PCT/US00/31247, particularly1-(2-aminopropyl)-3-methyl-1H-imdazol-6-ol fumarate and2-(3-chloro-6-methoxy-indazol-1-yl)-1-methyl-ethylamine. An example of ahypotensive lipid (the carboxylic acid group on the α-chain link of thebasic prostaglandin structure is replaced with electrochemically neutralsubstituents) is that in which the carboxylic acid group is replacedwith a C₁₋₆ alkoxy group such as OCH₃ (PGF_(2a) 1-OCH₃), or a hydroxygroup (PGF_(2a) 1-OH).

Preferred potassium channel blockers are calcium activated potassiumchannel blockers. More preferred potassium channel blockers are highconductance, calcium activated potassium (Maxi-K) channel blockers.Maxi-K channels are a family of ion channels that are prevalent inneuronal, smooth muscle and epithelial tissues and which are gated bymembrane potential and intracellular Ca²⁺.

The present invention is based upon the finding that maxi-K channels, ifblocked, inhibit aqueous humor production by inhibiting net solute andH₂O efflux and therefore lower IOP. This finding suggests that maxi-Kchannel blockers are useful for treating other ophthamologicaldysfunctions such as macular edema and macular degeneration. It is knownthat lowering IOP promotes blood flow to the retina and optic nerve.Accordingly, the compounds of this invention are useful for treatingmacular edema and/or macular degeneration.

It is believed that maxi-K channel blockers which lower IOP are usefulfor providing a neuroprotective effect. They are also believed to beeffective for increasing retinal and optic nerve head blood velocity andincreasing retinal and optic nerve oxygen by lowering IOP, which whencoupled together benefits optic nerve health. As a result, thisinvention further relates to a method for increasing retinal and opticnerve head blood velocity, increasing retinal and optic nerve oxygentension as well as providing a neuroprotective effect or a combinationthereof.

A number of marketed drugs function as potassium channel antagonists.The most important of these include the compounds Glyburide, Glipizideand Tolbutamide. These potassium channel antagonists are useful asantidiabetic agents. The compounds of this invention may be combinedwith one or more of these compounds to treat diabetes.

Potassium channel antagonists are also utilized as Class 3antiarrhythmic agents mid to treat acute infarctions in humans. A numberof naturally occurring toxins are known to block potassium channelsincluding Apamin, Iberiotoxin, Charybdotoxin, Noxiustoxin, Kaliotoxin,Dendrotoxin(s), mast cell degranuating (MCD) peptide, and β-Bungarotoxin(β-BTX). The compounds of this, invention may be combined with one ormore of these compounds to treat arrhythmias.

Depression is related to a decrease in neurotransmitter release. Currenttreatments of depression include blockers of neurotransmitter uptake,and inhibitors of enzymes involved in neurotransmitter degradation whichact to prolong the lifetime of neurotransmitters.

Alzheimer's disease is also characterized by a diminishedneurotransmitter release. Three classes of drugs are being investigatedfor the treatment of Alzheimer's disease cholinergic potentiators suchas the anticholinesterase drugs (e.g., physostigmine (eserine), andTacrine (tetrahydroaminocridine)); nootropics that affect neuronmetabolism with little effect elsewhere (e.g. Piracetam, Oxiracetam; andthose drugs that affect, brain vasculature such as a mixture of ergoloidmesylates and calcium channel blocking drugs including Nimodipine.Selegiline, a monoamine oxidase B inhibitor which increases braindopamine and norepinephrine has reportedly caused mild improvement insome Alzheimer's patients. Aluminum chelating agents have been ofinterest to those who believe Alzheimer's disease is due to aluminumtoxicity. Drugs that affect behavior, including neuroleptics, andanxiolytics have been employed. Anxiolytics, which are mildtranquilizers, are less effective than neuroleptics. The presentinvention is related to novel compounds which are useful as potassiumchannel antagonists.

The compounds within the scope of the present invention exhibitpotassium channel antagonist activity and thus are useful in disordersassociated with potassium channel malfunction. A number of cognitivedisorders such as Alzheimer's Disease, memory loss or depression maybenefit from enhanced release of neurotransmitters such as serotonin,dopamine or acetylcholine and the like. Blockage of Maxi-K channelsmaintains cellular depolarization and therefore enhances secretion ofthese vital neurotransmitters.

The compounds of this invention may be combined with anticholinesterasedrugs such as physostigmine (eserine) and Tacrine(tetrahydroaminocridine), nootropics such as Piracetam, Oxiracetam,ergoloid mesylates, selective calcium channel blockers such asNimodipine, or monoamine oxidase B inhibitors such as Selegiline, in thetreatment of Alzheimer's disease. The compounds of this invention mayalso be combined with Apamin, Iberiotoxin, Charybdotoxin, Noxiustoxin,Kaliotoxin, Dendrotoxin(s), mast cell degranuating (MCD) peptide,β-Bungarotoxin (β-BTX) or a combination thereof in treating arrythmias.The compounds of this invention may further be combined with Glyburide,Glipizide, Tolbutamide or a combination thereof to treat diabetes.

The herein examples illustrate but do not limit the claimed invention.Each of the claimed compounds are potassium channel antagonists and arethus useful in the described neurological disorders in which it isdesirable to maintain the cell in a depolarized state to achieve maximalneurotransmitter release. The compounds produced in the presentinvention are readily combined with suitable and known pharmaceuticallyacceptable excipients to produce compositions which may be administeredto mammals, including humans, to achieve effective potassium channelblockage.

For use in medicine, the salts of the compounds of formula I will bepharmaceutically acceptable salts. Other salts may, however, be usefulin the preparation of the compounds according to the invention or oftheir pharmaceutically acceptable salts. When the compounds of thepresent invention is acidic, suitable “pharmaceutically acceptablesalts” refers to salts prepared from pharmaceutically acceptablenon-toxic bases including inorganic bases and organic bases. Saltsderived from inorganic bases include aluminum, ammonium, calcium,copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous,potassium, sodium, zinc and the like. Particularly preferred are theammonium, calcium, magnesium, potassium and sodium salts. Salts derivedfrom pharmaceutically acceptable organic non-toxic bases include saltsof primary, secondary and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as arginine, betaine caffeine, choline,N,N¹-dibenzylethylenediamine, diethylamin, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine,hydrabamine, isopropylamine, lysine, methylglucamine, morpholine,piperazine, piperidine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine tripropylamine, tromethamineand the like.

When the compound of the present invention is basic, salts may beprepared from pharmaceutically acceptable non-toxic acids, includinginorganic and organic acids. Such acids include acetic, benzenesulfonic,benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic,glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic,mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic,phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid and thelike. Particularly preferred are citric, hydrobromic, hydrochloric,maleic, phosphoric, sulfuric and tartaric acids.

The preparation of the pharmaceutically acceptable salts described aboveand other typical pharmaceutically acceptable salts is more fullydescribed by Berg et al., “Pharmaceutical Salts,” J. Pharm. Sci.,1977:66:1-19.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specific amounts, aswell as any product which results, directly or indirectly, fromcombination of the specific ingredients in the specified amounts.

When a compound according to this invention is administered into a humansubject, the daily dosage will normally be determined by the prescribingphysician with the dosage generally varying according to the age,weight, sex and response of the individual patient, as well as theseverity of the patient's symptoms.

The maxi-K channel blockers used can be administered in atherapeutically effective amount intravenously, subcutaneously,topically, transdermally, parenterally or any other method known tothose skilled in the art.

Ophthalmic pharmaceutical compositions are preferably adapted fortopical administration to the eye in the form of solutions, suspension,ointments, creams or as a solid insert. Ophthalmic formulations of thiscompound may contain from 0.001 ppm to 1% and especially 0.1 ppm to 1%of medicament. Higher dosages as for example, about 10% or lower dosagescan be employed provided the dose is effective in reducing intraocularpressure, treating glaucoma, increasing blood flow velocity or oxygentension. For a single dose, from between 0.01 to 5000 ng, preferably 0.1to 500 ng, and especially 1 to 100 ng of the compound can be applied tothe human eye.

The pharmaceutical preparation which contains the compound may beconveniently admixed with a non-toxic pharmaceutical organic carrier, orwith a non-toxic pharmaceutical inorganic carrier. Typical ofpharmaceutically acceptable carriers are, for example, water, mixturesof water and water-miscible solvents such as lower alkanols oraralkanols, vegetable oils, polyalkylene glycols, petroleum based jelly,ethyl cellulose, ethyl oleate, carboxymethyl-cellulose,polyvinylpyrrolidone, isopropyl myristate and other conventionallyemployed acceptable carriers. The pharmaceutical preparation may alsocontain non-toxic auxiliary substances such as emulsifying, preserving,wetting agents, bodying agents and the like, as for example,polyethylene glycols 200, 300, 400 and 600, carbowaxes 1,000, 1,500,4,000, 6,000 and 10,000, antibacterial components such as quaternaryammonium compounds, phenylmercuric salts known to have cold sterilizingproperties and which are non-injurious in use, thimerosal, methyl andpropyl paraben, benzyl alcohol, phenyl ethanol, buffering ingredientssuch as sodium borate, sodium acetates, gluconate buffers, and otherconventional ingredients such as sorbitan monolaurate, triethanolamine,oleate, polyoxyethylene sorbitan monopalmitylate, dioctyl sodiumsulfosuccinate, monothioglycerol, thiosorbitol, ethylenediaminetetracetic acid, and the like. Additionally, suitable ophthalmicvehicles can be used as carrier media for the present purpose includingconventional phosphate buffer vehicle systems, isotonic boric acidvehicles, isotonic sodium chloride vehicles, isotonic sodium boratevehicles and the like. The pharmaceutical preparation may also be in theform of a microparticle formulation. The pharmaceutical preparation mayalso be in the form of a solid insert. For example, one may use a solidwater soluble polymer as the carrier for the medicament. The polymerused to form the insert may be any water soluble non-toxic polymer, forexample, cellulose derivatives such as methylcellulose, sodiumcarboxymethyl cellulose, (hydroxyloweralkyl cellulose), hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose;acrylates such as polyacrylic acid salts, ethylacrylates,polyactylamides; natural products such as gelatin; alginates, pectins,tragacanth, karaya, chondrus, agar, acacia; the starch derivatives suchas starch acetate, hydroxymethyl starch ethers, hydroxypropyl starch, aswell as other synthetic derivatives such as polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl methyl ether, polyethylene oxide, neutralizedcarbopol and xanthan gum, gellan gum, and mixtures of said polymer.

Suitable subjects for the administration of the formulation of thepresent invention include primates, man and other animals, particularlyman and domesticated animals such as cats and dogs.

The pharmaceutical preparation may contain non-toxic auxiliarysubstances such as antibacterial components which are non-injurious inuse, for example, thimerosal, benzalkonium chloride, methyl and propylparaben, benzyldodecinium bromide, benzyl alcohol, or phenylethanol;buffering ingredients such as sodium chloride, sodium borate, sodiumacetate, sodium citrate, or gluconate buffers; and other conventionalingredients such as sorbitan monolaurate, triethanolamine,polyoxyethylene sorbitan monopalmitylate, ethylenediamine tetraaceticacid, and the like.

The ophthalmic solution or suspension may be administered as often asnecessary to maintain an acceptable IOP level in the eye. It iscontemplated that administration to the mammalian eye will be about onceor twice daily.

For topical ocular administration the novel formulations of thisinvention may take the form of solutions, gels, ointments, suspensionsor solid inserts, formulated so that a unit dosage comprises atherapeutically effective amount of the active component or somemultiple thereof in the case of a combination therapy.

The following examples given by way of illustration is demonstrative ofthe present invention.

Definitions of the terms used in the examples are as follows:

SM—Starting material,

DMSO—dimethyl sulfoxide,

TLC—thin layer chromatography,

SGC—silica gel chromatography,

PhMgBr—phenylmagnesiumbromide

h=hr=hour.

THF—tetrahydrofuran,

DMF—dimethylformamide,

min—minute,

LC/MS—liquid chromatography/mass spectrometry,

HPLC—high performance liquid chromatography,

DBOP—Benzotriazol-1-yloxytris-(dimethyl amino)phosphoniumhexafluorophosphate,

equiv=eq=equivalent,

NBS—N-Bromosuccinamide and

AIBN-2,2′-azobisisobutyronitrile.

The compounds of this invention can be made, with modification whereappropriate, in accordance with Schemes 1 through 4. Examples 1-3 arealso produced in accordance with Schemes 1 and/or 2.

In Schemes 1 and 2 nitroanisole is brominated rising NBS, AIBN andbenzoyl peroxide. Treatment of the bromonitroanisole with potassiumcyanide yielded the cyanonitroanisole. Conversion of the nitro group toan amine is accomplished by hydrogenation. The amine is then treatedwith sodium nitrite and HCl to yield the indazole ring. In this reactionas soon as the diazonium is generated by nitrosation of the anilinemoiety it is trapped intramolecularily by the acidic benzyl cyanide.Tautomerization of the resultant derivative gives the indazole nucleus.Treatment of the nitrile with a Gringard followed by hydrolysis of thevesnltant imino-magnesium complex gives the desired alkyl/aryl ketone.

Preparative Example 1

In a 500 mL flask was charged 336 mmoles (13.44 g; 60%) of NaH. Underargon 150 mL of DMSO was added, followed by dropwise addition of 32 mLof ethyl cyanoacetate (2.2 equiv.; 352 mmoles) at 5° C. After al theaddition the reaction was warmed upto room temperature over 1 h. 30 g ofstarting nitro benzene derivative was added (160 mmoles) as a powder.The reaction mixture was heated in a closed system at 90° C. for 8hours. Acidification and standard work-up gave a crude oily residuewhich was purified over a silica-gel column to give 39 g of desiredcrystalline product which was decarboxylated to give the benzyl nitrileas follows. Thirty eight grams of SM obtained above was dissolved ins400 mL or 1N sodium carbonate. The homogenous solution was stirred at rtfor two days. TLC analysis indicated completion of reaction. Thereaction mixture was acidified and extracted with ethyl acetate (100mL×4). The combined organic phases was dried over sodium sulphate andconcentrated and residue was subjected to SGC to give the desiredproduct.

1H NMR CDCL3: 7.72 (1H, d, J=3 Hz); 7.61 (1H, d, J=8.5 Hz); 7.25 (1H,dd, J=3 and 8.5 Hz); 4.17 (2H, s); 3.94 (3H, s). LCMS [M+H]=193.

Preparative Example 2

10 g of benzylnitrile derivative was dissolved in THF 20 mL followed bydilution with 50 mL of methanol. The reaction mixture was taken in apressure tube, Pd—C

(10% wt/10 mole %) was added and the reaction mixture was hydrogenatedat 40 psi. After the requisite amount of hydrogen for the reduction ofthe NO₂ group was consumed the reaction was stopped. TLC analysisindicated a spot to spot conversion. The reaction mixture was filteredover a pad of celite and the filtrate was concentrated to a solid andused in the next step directly. Crude aniline derivative (52 mmoles wasdissolved/suspended in 2N HCl (150 mL), cooled to 5° C. followed by theaddition of 5.4 g of sodium nitrite in 10 mL of water. The reactionmixture was allowed to stir for 1 h with gradual warming to roomtemperature. TLC analysis indicated complete consumption of SM and theformation of a new spot. The reaction mixture was extracted with ethylacetate (100 mL×4); organic phase was collected, dried and concentrated.The residue was purified by SGC to give desired product. LCMS [M+H]=174

Preparative Example 3

Nitrile (1.5 g) obtained from Preparative Example 2 was dissolved in 20mL of dry THF and under argon 3 equiv. of PhMgBr (1M in THF) was addedat 5° C. The reaction mixture was stirred at room temperature for 1 h.The reaction was carefully quenched by addition of water and 1N HCl (15ml). The quenched reaction mixture was stirred at room temperature for 1hour then extracted with ethyl acetate (20 mL×3); combined organicphases were dried over sodium sulfate and concentrated to a solidresidue which was azeotroped with toluene three times.

LCMS [M+H]=253

Preparative Example 4

Weighed out 4.15 g of indazole and azeoptroped water with 2 toluene (100ml) washings, pulling off toluene azeotrope by rotovap. Dried thoroughlyunder high vacuum and performed argon purges. Dissolved in 40 ml dry THFand 92 ml dry ether under argon. Cooled to 5° C. in ice water bath.Charged 3 eq of isopropylmagnesium chloride ((6 ml of a 2M solution inTHF) and stirred for 0.5 hr at room temp. Carefully charged 1N HCl (240ml) and stirred for 1 h. Monitored reaction by TLC. Extracted withEtOAc, rotovaped and produced desired product.

LCMS [M+H]=219

Preparative Example 5

Step A:

100 g of 2-fluoro-4-methoxy-acetophenone in 400 mL of ethylene glycolwas stirred at room temperature with hydrazine (0.624 mol, 20 g) for 4 hafter which the reaction mixture was heated to 150° C. for 48 h. TLCanalysis indicated complete reaction. Partitioned the reaction mixtureinto dichloromethane and brine. Dried organic phase over sodium sulphateand evaporated to a solid. Re-crystallized from hexane/dicholomethanegave 6-methoxy-3-methyl-1H-indazole.

1H NMR (CDCL3): 7.5 (1H, d, 7.5 Hz); 6.8 (2H, m); 3.8 (3H, s); 2.55 (3H,s)

LCMS [M+H]=163

100 g of BOC-protected indazole was dissolved in 600 mL of CCl4,followed by addition of 1.1 equiv of NBS and 0.2 equiv of Bz2O. Reactionmix was vac-purged with argon and set to reflux for 5 h in presence oflight from a sun lamp. Reaction mixture was filtered over a pad of SGand concentrated. Residual oil was purified over a short SGC.Mono-bromide and mixed fractions of di-bromo derivative were obtained.

mono-bromide: 1H NMR (CDCL3): 7.7 (1H, d, 7.5 Hz); 7.6 (1H, bs); 6.95(1H, dd); 4.7 (2H, s); 3.9 (3H, s); 1.7 (9H, s);

di-bromide: 1H NMR (CDCL3): 8.05 (1H, d, J=7.5 Hz); 7.6 (1H, bs); 7.0(1H, dd); 6.85 (1H, s); 3.9 (3H, s); 1.7 (9H, s);

78 g of 6-methoxy-3-methyl-1H-indazole was dissolved in 1 L of MeCNcontaining 111 equiv of tri-ethyl amine, 0.2 equiv of DMAP was cooled to−5° C.; followed by slow addition of Boc₂O (1.1 equiv) in 200 mL ofMeCN. After 2 h of stirring the reaction at room temperature thereaction mixture was evaporated to an oil which was partitioned betweenEtOAc and brine, dried over sodium sulphate and evaporated. The residuewas applied to a short SGC and eluted with 15% EtOAc in hexane.Evaporation gave Boc-protected product.

1H NMR (CDCL3): 7.6 (1H, bs); 7.42 (1H, d, J=7.5 Hz); 6.85 (1H, dd); 3.8(3H, s); 2.5 (3H, s); 1.7 (9H, s)

LCMS [M+H]=263

To a solution of dibromide (23.2 g) in acetic acid was added sodiumacetate (22.5 g). The mixture was placed in oil bath and refluxed for acouple of hours until reaction completed. The mixture was cooled to roomtemperature and then poured into ice/water to give desired compound asan off-white solid. The solid was isolated by filtration and dried overnitrogen atmosphere.

¹H NMR (CDCl₃): δ 10.23 (1H, s); 8.19 (1H, d); 7.02 (1H, dd); 6.96 (1H,d); 3.90 (3H, s).

Step B:

To the intermediate from Step A was added triethyl orthoformate (40 ml)and heated to 130° C. for a couple of hours. The resulting mixture wasconcentrated to dry to give title compound as a brown solid.

¹H NMR (DMSO): δ 10.08 (1H, s); 7.98 (1H, d); 7.25 (1H, d); 7.02 (1H,dd); 6.81 (1H, s); 3.82 (3H, s); 3.52 (4H, q); 1.11 (6H, t).

Preparative Example 6

Oil free NaH (120 mg, 60% NaH in mineral oil was washed with hexanes 3times.) suspended in DMF was added intermediate from Preparative Example2 (346 mg) at RT. After bubbles subsided, the mixture was stirred at RTfor 30 min and MOM—Cl (0.23 ml) was added. After the reaction completed,the mixture was poured into ice/water to give compound as a solid. Thecrude material was purified by silica gel (hexanes/ethyl acetate=3/1) togive title compound.

¹H NMR (CDCl₃): 7.72 (1H, d); 7.06 (1H, dd); 6.99 (1H, d); 5.72 (2H, s);3.94 (3H, s); 3.36 (3H, s).

Preparative Example 7

To a solution of intermediate from preparative Example 2 (1.00 g, 5.75mmol) dissolved in THF (15 mL) was added cyclopentyl magnesium bromide(6.32 mL, 12.65 mmol) at 0° C. The reaction was allowed to warm toambient temperature and was quenched with saturated NH₄Cl uponcompletion. The resulting reaction mixture was extracted with EtOAc andthe combined organic layers were washed with brine, dried over MgSO₄,and concentrated in vacuo. The product was purified via SiO₂ gelchromatography to yield 580 mg of the desired product. ¹H NMR (CDCl₃) δ:1.702 (2H, m), 1.803 (2H, m), 2.005 (4H, m), 3.904 (3H, s), 4.070 (1H,m), 6.915 (1H, s), 7.010 (1H, d), 8.272 (1H, d).

Preparative Example 8

The desired compound was prepared by a procedure similar to the onedescribed for Preparative Example 7, but cyclohexyl magnesium bromidewas used in place of cyclopentyl magnesium bromide. ¹H NMR (CDCl₃) δ:1.321 (1H, m), 1.479 (2H, m), 1.604 (2H, m), 1.781 (1H, m), 1.861 (2H,m), 2.000 (2H, m), 3.641 (1H, m), 3.902 (3H, s), 6.923 (1H, s), 7.008(1H, d), 8.259 (1H, d).

Example 1

Indazole (0.55 mmoles from Preparative Example 3) starting materialobtained as above was dissolved in DMF (3 mL) followed by the additionof sodium hydride (0.88 mmoles). The reaction was stirred at roomtemperature for 15 min, followed by the addition of 1-bromo-pinacolone(0.669 mmoles). The reaction was stirred at room temperature for 30 min.TLC and LC-MS analysis indicated complete consumption of startingmaterial concurrent with the formation of a new product spot. Thereaction mixture was quenched by the addition of water. Standard aqueouswork-up followed by purification of crude by SGC gave the desiredproduct as white solid.

1H NMR CDCL3: 8.3 (3H, m); 7.61 (1H, t, J=7.5 Hz); 7.52 (2H, dd, J=7.5and 7.0 Hz); 7.04 (1H, dd, J=2 and 9 Hz); 6.56 (1H, d, J=2 Hz); 5.4 (2H,s); 3.94 (3H, s); 1.4 (9H, s). LCMS [M+H]=351.

Example 2

Indazole (0.60 mmoles from Preparative Example 4) starting materialobtained as above was dissolved in DMF (3 mL) followed by the additionof sodium-hydride (0.88 mmoles). The reaction was stirred at roomtemperature for 15 min, followed by the addition of 1-bromo-pinacolone(0.669 mmoles). The reaction was stirred at room temperature for 30 min.TLC and LC-MS analysis indicated complete consumption of startingmaterial concurrent with the formation of a new product spot. Thereaction mixture was quenched by the addition of water. Standard aqueouswork-up followed by purification of crude by SGC gave the desiredproduct as white solid.

1H NMR in CDCL: 8.22 (1H, d, J=9 Hz); 6.97 (1H, dd, J=2 and 9 Hz); 6.5(1H, d J=2 Hz); 5.4 (2H, s); 3.94 (3H, s); 2.8 (1H, m); 1.38 (9H, s);1.27 (6H, d, J=6.5 Hz).

LCMS=[M+H]=317

Example 3

133 mg of indazole from Preparative Example 4 was dissolved in dry DMF(3 mL), followed by the addition of sodium hydride (24.3 mg, 60% oildispersion). After stirring at room temperature for 15 min. 0.2 mL of2-ethyl-hexyl iodide was added. The reaction mixture was allowed to stirfor an additional 10 h. Upon standard aqueous work-up followed bypurification by SGC the desired product was obtained.

1HNMR CDCL3: 8.22 (1H, d, J=8.5 Hz); 7.0 (1H, dd, J=8.5 and 2 Hz); 6.75(1H, d, J=2 Hz); 4.23 (2H, d, J=7.5 Hz); 3.9 (3H, s); 2.2 (1H, m);0.8-1.5 (15h, m).

LCMS [M+H]=331

Examples 4 through 15 as shown below are made, with some modification ofthe desired compound of Example 3, by alkylation of the indazole asdescribed in Example 1. Additionally, analogs of Examples 1 and 4-15 canbe prepared following analogous procedures using the indazole ofPreparative Example 4 or alternatively another indazole preparedfollowing procedures described herein.

Example 4

1H NMR CDCL3: 8.35 (3H, m); 7.6 (1H, t); 7.55 (2H, t); 7.1 (1H, dd,J=8.5 and 2 Hz); 6.8 (1H, d, J=2 Hz); 5.9 (1H, m); 5.15 (2H, m); 4.5(2H, t); 3.9 (3H, s); 2.8 (2H, m).

LCMS [M+H]=307

Example 5

1H NMR CDCL3: 8.35 (3H, m); 7.6 (1H, t); 7.55 (2H, t); 7.1 (1H, dd,J=8.5 and 2 Hz); 6.8 (1H, d, J=2 Hz); 4.25 (2H, d, J=7.5 Hz); 3.9 (3H,s); 1-2.2 (11H, m).

LCMS [M+H]=349

Example 6

1H NMR CDCL3: 8.35 (3H, m); 7.6 (1H, t); 7.55 (2H, t); 7.1 (1H, dd,J=8.5 and 2 Hz); 6.8 (1H, d, J=2 Hz); 4.45 (2H, d, J=7.5 Hz); 3.9 (3H,s); 3.0 (1H, m); 1.8-2.2 (6H, m).

LCMS [M+H]=321

Example 7

1H NMR CDCL3: 8.35 (3H, m); 7.6 (1H, t); 7.55 (2H, t); 7.1 (1H, dd,J=8.5 and 2 Hz); 6.8 (1H, d, J=2 Hz); 4.35 (2H, d, J=7.5 Hz); 3.9 (3H,s); 1.4 (1H, m); 0.7 (2H, m); 0.5 (2H, m).

LCMS [M+H]=307

Example 8

1H NMR CDCL3: 8.35 (3H, m); 7.6 (1H, t); 7.55 (2H, t); 7.1 (1H, dd,J=8.5 and 2 Hz); 6.8 (1H, d, J=2 Hz); 4.2 (3H, s); 3.9 (3H, s); 1.1 (9H,s).

LCMS [M+H]=323

Example 9

1H NMR CDCL3: 8.35 (3H, m); 7.6 (1H, t); 7.55 (2H, t); 7.1 (1H, dd,J=8.5 and 2 Hz); 6.8 (1H, d, J=2 Hz); 4.25 (2H, d, J=7.5 hZ); 3.9 (3H,s); 2.6 (1H, m); 1.02 (6H, d).

LCMS [M+H]=309

Example 10

1H NMR CDCL3: 8.35 (3H, m); 7.6 (1H, t); 7.55 (2H, t); 7.1 (1H, dd,J=8.5 and 2 Hz); 6.8 (1H, d, J=2 Hz); 4.4 (2H, t, J=7.5 Hz); 3.9 (3H,s); 2.o (2H, m); 1.02 (3H, t, J=7.5 Hz).

LCMS [M+H]=295

Example 11

1H NMR CDCL3: 8.35 (3H, m); 7.6 (1H, t); 7.55 (2H, t); 7.1 (1H, dd,J=8.5 and 2 Hz); 6.8 (1H, d, J=2 Hz); 4.4 (2H, t, J=7.5 Hz); 3.9 (3H,s); 2.0 (2H, m); 0.8-1.5 (5H, m).

LCMS [M+H]=337

Example 12

1H NMR CDCL3: 8.35 (3H, m); 7.6 (1H, t); 7.55 (2H, t); 7.1 (1H, dd,J=8.5 and 2 Hz); 6.8 (1H, d, J=2 Hz); 6.2 (1H, m); 5.0-5.4 (3H, m); 3.9(3H, s).

LCMS [M+H]=293

Example 13

1H NMR CDCL3: 8.35 (3H, m); 7.6 (1H, t); 7.55 (2H, t); 7.1 (1H, dd,J=8.5 and 2 Hz); 6.8 (1H, d, J=2 Hz); 4.4 (2H, d, J=7.5 Hz); 3.9 (3H,s); 2.1 (1H, m); 1.4 (4H, m); 1.0 (6H, t, J=7.5 Hz).

LCMS [M+H]=337

Example 14

1H NMR CDCL3: 8.35 (3H, m); 7.6 (1H, t); 7.55 (2H, t); 7.1 (1H, dd,J=8.5 and 2 Hz); 6.8 (1H, d, J=2 Hz); 4.4 (2H, t, J=7.5 Hz); 3.9 (3H,s); 1.9 (2H, t, J=7.5 Hz); 1.1 (9H, s).

LCMS [M+H]=337.

Example 15

1H NMR CDCL3: 8.35 (3H, m); 7.6 (1H, t); 7.55 (2H, t); 7.1 (1H, dd,J=8.5 and 2 Hz); 6.8 (1H, d, J=2 Hz); 4.5 (2H, t, J=7.5 Hz); 3.9 (3H,s); 1.9 (2H, m); 1.7 (1H, m); 1.05 (6H, d, J=7.5 Hz).

LCMS [M+H]=323

Example 16

Step A:

To a solution of 5-iodo-2-chloropyridine (2.56 g, 10.78 mmol) in THF (10mL) was added iPrMgBr dropwise at −78° C. The reaction stirred for 1 hbefore Preparative Example 5 (1.17 g, 6.10 mmol) was added as a solutionin THF (5 mL). After 2 h and the reaction was quenched with 1N NaOH andextracted with EtOAc. The combined organic layers were washed withbrine, dried over MgSO₄, and concentrated in vacuo. To a solution of thecrude product in toluene (50 mL) was added MnO₂ (2.173 g, 25.0 mmol) andthe reaction mixture was heated to 130° C. After 1 h the reaction wascomplete, filtered through a celite pad, and concentrated in vacuo. Thecrude product was dissolved in THF (16 mL) and 4 mL of 1N HCl was addeddropwise. The reaction stirred at RT until TLC analysis indicatedcompletion. The reaction mixture was cooled to 0° C. and the solidprecipitate was collected. ¹H NMR (CD₃OD) δ: 3.900 (3H, s), 7.013 (1H,d), 7.062 (1H, s), 7.627 (1H, d), 8.672 (1H, d), 9.306 (1H, s).

Step B:

To a solution of the intermediate from Step A (1.00 g, 3.48 mmol) andCs₂CO₃ (3.396 g, 10.45 mmol) in DMF (14 mL) was added 1-chloropinacolone(0.681 mL, 5.22 mmol). After 40 min the reaction was complete andquenched with H₂O. The reaction mixture was extracted with EtOAc and thecombined organic layers were washed with H₂O, brine dried over MgSO₄,and concentrated in vacuo to yield the desired product. ¹H NMR (CD₃OD)δ: 1.344 (9H, s), 3.888 (3H, s), 6.947 (1H, s), 7.043 (1H, d), 7.625(1H, d), 8.221 (1H, d), 8.624 (1H, d), 9.257 (1H, d).

Step C:

40.6 mg (1.036 mmol) of NaH (60% dispersion in mineral oil) was washed3× with hexane and dried under nitrogen. Ethylene glycol (1 mL) wasadded to the dry NaH and the reaction stirred for 20 min at 60° C. Tothe reaction mixture was added the intermediate from Step B (100 mg,0.259 mmol) as a solution in THF (1.5 mL). The reaction continued tostir overnight at 60° C. Upon completion, the THF was removed in vacuo,diluted with EtOAc, washed with H₂O, brine, dried over MgSO₄, andconcentrated in vacuo. The crude residue was purified via silica gelchromatography.

1.376 (9H, s), 3.889 (3H, s), 4.021 (2H, m), 4.608 (2H, m), 5.429 (2H,s), 6.543 (1H, s), 6.223 (1H, d), 7.054 (1H, d), 8.336 (1H, d), 8.541(1H, d), 9.310 (1H, s).

Example 17

Step A:

To a solution of 5-bromo-2-methylpyridine (736 mg, 4.31 mmol) in THF (15mL) was added nBuLi dropwise (2.156 mL, 5.39 mmol, 2.5 M in hexanes) at−78° C. The reaction stirred for 1 h before Preparative Example 5 (1.00g, 3.59 mmol) was added as a solution in THF (5 mL). The startingmaterial was consumed after 2 h and the reaction was quenched with 1NNaOH and extracted with EtOAc. The combined organic layers were washedwith brine, dried over MgSO₄, and concentrated in vacuo. A solution ofthe crude product in toluene (20 mL) was added MnO₂ (0.414 g, 4.77 mmol)and the reaction mixture was heated to 130° C. After 1 h the reactionwas complete, filtered through a celite pad, and concentrated in vacuo.The crude product was dissolved in THF and 4 mL of 1N HCl was addeddropwise. After 1 h reaction mixture was cooled to 0° C. and the solidprecipitate was collected. ¹H NMR (DMSO) δ: 2.553 (3H, s), 3.832 (3H,s), 7.000 (1H, d), 7.089 (1H, s), 7.451 (1H, d), 8.100 (1H, d), 8.430(1H, d), 9.220 (1H, s).

Step B:

This compound was made as described in Step B of Example 16.

¹H NMR (CDCl₃) δ: 1.38 (9H, s), 2.65 (3H, s), 3.85 (3H, s), 5.22 (2H,s), 6.56 (1H, s), 7.05 (1H, d), 7.32 (1H, d), 8.34 (1H, d), 8.45 (1H,d), 9.50 (1H, s).

Step C:

To a stirring solution of the intermediate from Step B (74 mg, 0.202mmol) in CH₂Cl₂ was added MCPBA (67 mg, 0.303 mmol) at 0° C. TLCindicated the reaction was complete after 1.5 h and the reaction mixturewas concentrated in vacuo. The crude residue was dissolved in EtOAc andwashed with saturated sodium bisulfite, H₂O, brine, dried over MgSO₄,and concentrated in vacuo. Purified via silica gel chromatography. TheN-oxide was dissolved in CH₂Cl₂ and TFAA was added dropwise at 0° C.After 2 h the reaction was concentrated in vacuo and purified via silicagel chromatography.

¹H NMR (CDCl₃) δ: 1.373 (9H, s), 3.898 (3H, s), 4.882 (2H, s), 5.428(2H, s), 6.564 (1H, s), 7.066 (1H, d), 7.429 (1H, d), 8.352 (1H, d),8.581 (1H, d), 9.541 (1H, s).

Example 18

Step A:

To a solution of 2-pyridineacetic acid, 5-bromo-α,α-difluoro-, ethylester (13.4 g; prepared according to “Ero, H.; Haneko, Y.; Sakamoto, T.Chem Pharm. Bull. 2000, 48, 982.”) in ethanol was added sodiumborohydride (2.3 g) portion-wise at 0° C. After stirring at 0° C. for 1hour, the mixture was poured into water and extracted with ethylacetate. The organic layer was washed with 1N NaOH_(aq), brine, dried(MgSO₄), and concentrated under reduced pressure to afford crudealcohol. The crude alcohol in methylene chloride was added imidazole(4.1 g) and TBS—Cl (8.3 g) at 0° C. The mixture was stirred for 1 hour.The reaction was poured into 0.1 N HCl_(aq) extracted with methylenechloride. The organic layer was washed with brine, dried (MgSO₄) andevaporated. The residue was purified by silica gel (100% methylenechloride) to give desired compound as a colorless oil.

¹H NMR (CDCl₃): δ 8.75 (1H, d); 7.95 (1H, dd); 7.57 (1H, d); 4.20 (2H,t); 0.82 (9H, s); 0.02 (6H, s).

Step B:

The desired compound was prepared by a procedure similar to the onedescribed for Example 16, Step A.

¹H NMR (DMSO): δ 9.35 (1H, d); 8.65 (1H, dd); 8.14 (1H, d); 7.88 (1H,d); 7.10 (1H, d); 7.03 (1H, dd); 4.05 (2H, t); 3.85 (3H, s).

LC-MS (M+H)=334.2.

Step C:

The desired compound vas prepared by a procedure similar to the onedescribed for Example 16, Step B. This compound was purified by silicagel (hexanes/ethyl acetate=1/1) and crystallized from hexanes/ethylacetate.

¹H NMR (CHCl₃): δ 9.53 (1H, d); 8.71 (1H, dd); 8.35 (1H, d); 7.88 (1H,d); 7.08 (1H, dd); 6.57 (1H); 5.44 (2H, s); 4.32 (2H, t); 3.91 (3H, s);1.38 (9H, s).

LC-MS (M+H)=432.3.

Example 19

Step A:

To a solution of 2,5-dibromopyridine (2.4 g) in toluene was addedtributylallyltin (3.4 ml) and dichlorobis(triphenylphosphine)palladium(0.7 g) under nitrogen atmosphere. The mixture was refluxed for a coupleof hours and concentrated under reduced pressure. The residue wasre-dissolved in “wet ether” and added DBU (3 ml) slowly to give a cloudysolution. The mixture was filtered over a pad of silica gel andconcentrated. The residue was dissolved in methylenechloride/methanol=1/1 solution and cooled to −78° C. To this solutionwas bubbled though ozone until the reaction mixture became a blue color.The reaction was warmed to 0° C. and added sodium borohydride (0.5 g)portion-wise. After stirring at 0° C. for 1 hour, the mixture was pouredinto water and extracted with ethyl acetate. The organic layer waswashed with 1N NaOH_(aq), brine, dried (MgSO₄), and concentrated underreduced pressure to afford crude alcohol. The alcohol was purified bysilica gel (methylene chloride/ethyl acetate=1/1) to give desiredalcohol. To a solution of alcohol in methylene chloride was addedimidazole (0.4 g) and TBS—Cl (0.8 g) at 0° C. The mixture was stirredfor 1 hour. The reaction was poured into 0.1 N HCl_(aq) extracted withmethylene chloride. The organic layer was washed with brine, dried(MgSO₄) and evaporated. The residue was purified by silica gel (100%methylene chloride) to give desired compound.

¹H NMR (CDCl₃): δ 8.61 (1H, d); 7.73 (1H, dd); 7.14 (1H, d); 3.97 (2H,t); 2.96 (2H, t); 0.36 (9H, s); −0.02 (6H, s).

Step B:

The desired compound was prepared by a procedure similar to the onedescribed for Example 16, Steps A and B. This compound was purified bysilica gel (hexanes/ethyl acetate=1/3).

¹H NMR (CHCl₃): δ 9.53 (1H, d); 8.54 (1H, dd); 8.35 (1H, d); 7.37 (1H,d); 7.07 (1H, dd); 6.56 (1H, d); 5.45 (2H, s); 4.11 (2H, t); 3.90 (3H,s); 3.18 (2H, t); 1.38 (9H, s).

LC-MS (M+H)=396.2.

Example 20

Step A:

To a solution of chloroiodopyridine (1.2 g), pyrrolidine (1.2 ml) andpotassium carbonate (2.0 g) in DMF was heated 130° C. for 16 h. Themixture was cooled to RT and poured into ice/water to give crude solidmaterial. The title compound was crystallized from hexanes/ethyl acetate(0.73 g).

¹H NMR (CDCl₃): 8.30 (1H, d); 7.62 (1H, dd); 6.23 (1H, d); 3.43 (4H, m);2.03 (4H, m).

Step B:

To a solution of intermediate from Step A (274 mg) in THF was addedisopropyl magnesium chloride (0.5 ml, 2N in diethyl ether) at −78° C.The mixture was warmed up to RT for a couple hours to completeiodide-magnesium exchange and re-cooled to −78° C. when PreparativeExample 5 (110 mg) was added to the reaction mixture. The resultingsolution was stirred at RT for 16 h and quenched with 1N NaOH, exactedwith EtOAc. The organic layer was washed with brine, dried overmagnesium sulfate and evaporated under vacuum. The residue was dissolvedin 96% formic acid and heated to 100° C. for 16 h. After cooled to RT,the mixture was diluted with 1N NaOH to pH=9. The mixture was exactedwith EtOAc, brine, dried over magnesium sulfate and evaporated undervacuum to give crude material.

Step C:

The title compound was prepared as described in Preparative Example 6,using chlorepinacolone instead of MOM—Cl. The final compound waspurified by silica gel (hexanes/ethyl acetate 1/1).

¹H NMR (CDCl₃): 9.46 (1H, d); 8.40 (1H, d); 8.32 (1H, d); 7.00 (1H, dd);6.53 (1H, d); 6.48 (1H, d); 5.43 (2H, s); 3.89 (3H, s); 3.63 (4H, br,s); 2.08 (4H, br, s); 1.37 (9H, s). LCMS (M+H)=421.4.

Example 21

Step A: The title compound was prepared by a procedure similar to theone described for Example 20, Step A. The reaction used dimethylaminehydrogen chloride and potassium carbonate instead of pyrrolidine.

¹H NMR (CDCl₃): 8.31 (1H, d); 7.64 (1H, dd); 6.37 (1H, d); 3.08 (6H, s).

Step B:

The title compound was prepared by a procedure similar to the onedescribed for Example 20, Step B and C by Using intermediate fromExample 21, Step A instead of Example 20, Step A.

¹H NMR (CDCl₃): 9.41 (1H, d); 8.41 (1H, dd); 8.32 (1H, d); 7.01 (1H,dd); 6.60 (1H, d); 6.53 (1H, d); 5.42 (2H, s); 3.89 (3H, s); 3.24 (6H,s); 1.37 (9H, s).

LCMS (M+H)=395.4.

Example 22

To a solution of intermediate from Example 21, Step A in THF was addedisopropyl magnesium chloride (2N in diethyl ether) at −78° C. Themixture was warmed up to RT for a couple hours to completeiodide-magnesium exchange and re-cooled to −78° C. when PreparativeExample 6 (110 mg) was added to the reaction mixture. The resultingsolution was stirred at RT for 16 h and quenched with 1N NaOH, exactedwith EtOAc. The organic layer was washed with brine, dried overmagnesium sulfate and evaporated under vacuum. The title compound waspurified by silica gel (methylene chloride/ethyl acetate=10/1).

¹H NMR (CDCl₃): 9.46 (1H, d); 8.45 (1H, dd); 8.31 (1H, d); 7.04 (1H,dd); 6.98 (1H, d); 6.62 (1H, d); 5.77 (2H, s); 3.94 (3H, s); 3.39 (3H,s); 3.25 (4H, s);

LCMS (M+H)=342.2.

Example 23

Step A:

To a solution of 2-chloro-5-iodopyridine in THF was added isopropylmagnesium chloride (0.5 ml, 2N in diethyl ether) at −78° C. The mixturewas warmed up to RT for a couple hours to complete iodide-magnesiumexchange and re-cooled to −78° C. when Intermediate from PreparativeExample 2 was added to the reaction mixture. The resulting solution wasstirred at RT for 16 h and quenched with 1N NaOH, exacted with EtOAc.The organic layer was washed with brine, dried over magnesium sulfateand evaporated under vacuum.

Step B:

The title compound was prepared as described in Preparative Example 6,using chloropinacolone instead of MOM—Cl. The final compound waspurified by silica gel (hexanes/ethyl acetate=3/1).

¹H NMR (CDCl₃): 9.41 (1H, d); 8.53 (1H, dd); 8.33 (1H, d); 7.49 (1H,dd); 7.07 (1H, dd); 6.58 (1H, d); 5.43 (2H, s); 3.91 (3H, s); 1.37 (9H,s).

LCMS (M+H)=386.3.

Example 24

To a solution of intermediate from Example 23 in THF was added sodiummethoxide (3 eq. 25% in methanol). The mixture was refluxed untilreaction completed and quenched with 1N HCl. The mixture was extractedwith ethyl acetate, brine, dried over magnesium sulfate and evaporatedunder vacuum. The residue was purified with silica gel (hexanes/ethylacetate=2/1).

¹H NMR (CDCl₃): 9.35 (1H, d); 8.50 (1H, dd); 8.33 (1H, d); 7.04 (1H,dd); 6.86 (1H, d); 6.55 (1H, d); 5.43 (2H, s); 4.06 (3H, s); 3.90 (3H,s); 1.38 (9H, s).

LCMS (M+H)=386.3.

Example 25

To 195 mg of NaH (60% dispersion in oil washed with hexane) was addedDMF (10 mL) and Preparative Example 7 (597 mg, 2.44 mmol). The reactionstirred at room temperature for 30 min before 1-chloropinacolone (3.81mL, 2.92 mmol) was added. After 20 min the reaction was quenched withH₂O and diluted with EtOAc. The aqueous layer was extracted with EtOAcand the combined organic layers were washed with H₂O, brine, dried overMgSO₄, and concentrated in vacuo. The crude material was purified viasilica gel chromatography to yield.

¹H NMR (CDCl₃) δ: 1.361 (9H, s), 1.683 (2H, m), 1.788 (2H, m), 1.974(4H, m), 3.872 (3H, s), 4.029 (1H, m), 5.372 (2H, s), 6.514 (1H, s),6.986 (1H, d), 8.267 (1H, d).

Example 26

Using Preparative Example 8, this compound was prepared as described inExample 25. The title compound was purified via SiO₂ preparatory platechromatography. ¹H NMR (CDCl₃) δ: 1.285-1.575 (15H, m), 1.833 (2H, d),1.994 (2H, d), 3.615 (1H, m), 3.860 (3H, s), 5.372 (2H, s), 6.490 (1H,s), 6.981 (1H, d), 8.254 (1H, d).

Example 27

Using the intermediate from Example 23, this compound was prepared asdescribed in Example 16, Step C but 2-methoxy-ethanol was used in placeof ethylene glycol. The title compound was purified via SiO₂ preparatoryplate chromatography. ¹H NMR (CDCl₃) δ: 1.371 (9H, s), 3.475 (3H, s),3.802 (2H, t), 3.889 (3H, s), 4.620 (2H, t), 5.424 (2H, s), 6.542 (1H,s), 6.923 (1H, d), 7.043 (1H, d), 8.337 (1H, d), 8.501 (1H, d), 9.299(1H, s).

Example 28

di-tert-butyl4-{[1-(3,3-dimethyl-2-oxobutyl)-6-methoxy-1-H-indazole-3-yl]carbonyl}hydroxylEthyl Benzyl

100 g of fluoro-acetophenone in 400 mL of ethylene glycol was stirred atroom temperature with hydrazine (0.624 mol, 20 g) for 4 h after whichthe reaction mixture was heated to 150 C for 48 h. TLC analysisindicated complete reaction. Partitioned the reaction mixture intodichloromethane and brine. Dried organic phase over sodium sulphate andevaporated to a solid. Re-crystallized from hexane/dicholomethane gaveindazole.

1H NMR (CDCL3): 7.5 (1H, d, 7.5 Hz); 6.8 (2H, m); 3.8 (3H, s); 2.55 (3H,s)

LCMS [M+H]=163

6-methoxy-3-methyl-1-Htert-butyl-6-methoxy-3-methyl-1-indazole-carboxylate Indazole

78 g of indazole was dissolved in 1 L of MeCN containing 1.1 equiv oftri-ethyl amine, 0.2 equiv of DMAP was cooled to −5 C; followed by slowaddition of Boc2O (1.1 equiv) in 200 mL of MeCN. After 2 h of stirringthe reaction at rt the reaction mixture was evaporated to an oil whichwas partitioned between EtOAc and brine, dried over sodium sulphate andevaporated. The residue was applied to a short SGC and eluted with 15%EtOAc in hexane. Evaporation gave product.

1H NMR (CDCL3): 7.6 (1H, bs); 7.42 (1H, d, J=7.5 Hz); 6.85 (1H, dd); 3.8(3H, s); 2.5 (3H, s); 1.7 (9H, s)

LCMS [M+H]=263

100 g of indazole was dissolved in 600 mL of CCl4, followed by additionof 1.1 equiv of NBS and 0.2 equiv of Bz2O. Reaction mix was vac-purgedwith argon and set to reflux for 5 h in presence of light from a sunlamp. Reaction mixture was filtered over a pad of SG and concentrated.Residual oil was purified over a short SGC. 85 g of pure bromide wasobtained. Mixed fractions yielded di-bromo derivative

mono-bromide: 1H NMR (CDCL3): 7.7 (1H, d, 7.5 Hz); 7.6 (1H bs); 6.95(1H, dd); 4.7 (2H, s); 3.9 (3H, s); 1.7 (9H, s);

di-bromide: 1H NMR (CDCL3): 8.05 (1H, d, J=7.5 Hz); 7.6 (1H, bs); 7.0(1H, dd); 6.85 (1H, s); 3.9 (3H, s); 1.7 (9-H, s);

5 g of bromide was dissolved in 10 mL of DMSO, cooled to 0 C followed byaddition of 2.5 equiv of TMANO (trimethyl amine N-oxide). Reaction wasstirred for 0.5 h then a standard work-up and SG pad filteration gavedesired product quantitatively. LCMS [M+H]=277

1H NMR (CDCL3): 10.2 (1H, s); 8.1 (1H, d, J=7.5 Hz); 7.6 (1H, bs); 7.0(1H, dd); 3.9 (3H, s); 1.7 (9H, s);

Glasswares were Flame Dried Under High Vacuum

To neat iodo-benzyl alcohol derivative (3.6 g, 110 mmol) in the flaskwas slowly added isopropylMgCl (5 mL, 2M solution). After stirring at rtfor 2 hr, indazole derivative (1.1 g, 4 mmol) in 15 mL THF was added.The reaction mixture was stirred at rt for 2 hr. LC-MS showed thereaction was complete. Pour the reaction mixture into 30 mL saturatedNH4Cl, followed by adding 40 mL ether. The organic layer was separated,the aqueous layer was extracted by ether (40 mL). The combined organiclayers were washed with saturated K2CO3 (2×30 mL), water (40 mL) andbrine (20 mL). The solvent was removed, the residue was used for nextstep reaction without further purification. LCMS [M+H]=499

To a solution of indazole (crude from step 5) in 20 mL dichloromethanewas added 5 g celite and 4.3 g of PCC (MW 215.56, ˜2 eq). The reactionmixture was stirred at rt for 2 hr. LC-MS showed the reaction wascompleted LCMS [M+H]=497. The reaction mixture was filtered.

The solvent was removed, the residue was dissolved in 10 mL MeOH, andadded 20 mL 2N HCl. After stirring for 1 hr at rt LCMS and TLC analysisindicated complete reaction. The reaction mixture was extracted withEtOAc (2×30 mL). The solvent was removed, the residue was used for nextstep reaction without further purification. LCMS [M+H]=283

To a solution of indazole (342 mg crude prod from step 7, ˜10 mmol)) in15 mL, acetone was added 1.5 g of K2CO3 and 1.5 mL Bromopinacolone(Mw179.06, d1.326, 2.0 g, 11 mmol). The reaction mixture was stirred at80° C. in a sea) tube for 2 hr. After filtered off salts, the solventwas removed, the residue was purified by HPFC to give white solidproduct.

1H NMR (CDCL3)=8.3 (3H, m); 7.5 (1H, d, J=7.5 Hz); 7.05 (1H, dd); 7.6(1H, bs); 5.4 (2H, s); 4.8 (2H, bs); 3.9 (3H, s); 1.38 (9H, s)

LCMS [M+H]=381

Functional Assays

A. Maxi-K Channel

The identification of inhibitors of the Maxi-K channel can beaccomplished using Aurora Biosciences technology, and is based on theability of expressed Maxi-K channels to set cellular resting potentialafter transient transfection of both α and β subunits of the channel inTsA-201 cells. In the absence of inhibitors, cells display ahyperpolarized membrane potential, negative inside, close to E_(K) (−80mV) which is a consequence of the activity of the Maxi-K channel.Blockade of the Maxi-K channel will cause cell depolarization. Changesin membrane potential can be determined with voltage-sensitivefluorescence resonance energy transfer (FRET) dye pairs that use twocomponents, a donor coumarin (CC₂DMPE) and an acceptor

oxanol (DiSBAC₂(3)). Oxanol is a lipophilic anion and distributes acrossthe membrane according to membrane potential. Under normal conditions,when the inside of the cell is negative with respect to the outside,oxanol is accumulated at the outer leaflet of the membrane andexcitation of coumarin will cause FRET to occur. Conditions that lead tomembrane depolarization will cause the oxanol to redistribute to theinside of the cell, and, as a consequence, to a decrease in FRET. Thus,the ratio change (donor/acceptor) increases after membranedepolarization.

Transient transfection of the Maxi-K channel in TsA-201 cells can becarried out as previously described (Hanner et al. (1998) J. Biol. Chem.273, 16289-16296) using FUGENE6™ as the transfection reagent. Twentyfour hours after transfection, cells are collected in Ca²⁺—Mg²⁺-freeDulbecco's phosphate-buffered saline (D-PBS), subjected tocentrifugation, plated onto 96-well poly-d-lysine coated plates at adensity of 60,000 cells/well, and incubated overnight. The cells arethen washed 1× with D-PBS, and loaded with 100 μl of 4 μM CC₂DMPE-0.02%pluronic-127 in D-PBS. Cells are incubated at room temperature for 30min in the dark. Afterwards, cells are washed 2× with D-PBS and loadedwith 100 μl of 6 μM DiSBAC₂(3) in (mM): 140 NaCl, 0.1KCl, 2CaCl₂, 1MgCl,0 Hepes-NaOH, pH, 7.4, 10 glucose. Test compounds are diluted into thissolution, and added it the same time. Cells are incubated at roomtemperature for 30 min in the dark.

Plates are loaded onto a voltage/ion probe reader (VIPR) instrument, andthe fluorescene emission of both CC₂DMPE and DiSBAC₂(3) are recorded for10 sec. At this point, 100 μl of high-potassium solution (mM): 140KCl,2CaCl₂, 1MgCl₂, 20 Hepes-KOH, pH 7.4, 10 glucose are added and thefluorescence emission of both dyes recorded for additional 10 sec. Theratio CC₂DMPE/DiSBAC₂(3), before addition of high-potassium solutionequals 1. In the absence of any inhibitor, the ratio after addition ofhigh-potassium solution varies between 1.65-2.0. When the Maxi-K channelhas been completely inhibited by either a known standard of testcompound, this ratio remains at 1. It is possible, therefore, to titratethe activity of a Maxi-K channel inhibitor by monitoring theconcentration-dependent change in the fluorescence ratio.

The compounds of this invention were found to causeconcentration-dependent inhibition of the fluorescence ratio with IC₅₀'sin the range of about 1 nM to about 20 μM, more preferably from about 10nM to about 500 nM.

B. Electrophysiological Assays of Compound Effects on High-ConductanceCalcium-Activated Potassium Channels

Human Non-Pigmented Ciliary Epithelial Cells

The activity of high-conductance calcium-activated potassium (maxi-K)channels in human non-pigmented ciliary epithelial cells was determinedusing electrophysiological methods. Currents through maxi-K channelswere recorded in the inside-out configuration of the patch clamptechnique, where the pipette solution faces the extracellular side ofthe channel and the bath solution faces the intracellular side. Excisedpatches contained one to about fifty maxi-K channels. Maxi-K channelswere identified by their large single channel conductance (250-300 pS),and by sensitivity of channel gating to membrane potential andintracellular calcium concentration. Membrane currents were recordedusing standard electrophysiological techniques. Glass pipettes (Garner7052) were pulled in two stages with a Kopf puller (model 750), andelectrode resistance was 1-3 megohms when filled with saline. Membranecurrents were recorded with EPC9 (HEKA Instruments) or Axopatch 1D (AxonInstruments) amplifiers, and digital conversion was done with ITC-16interfaces (Instrutech Corp). Pipettes were filled with (mM); 150KCl, 10Hepes, 1MgCl₂, 0.01CaCl₂, 3.65KOH, pH 7.20. The bath (intracellular)solution was identical, except, in some cases, calcium was removed, 1 mMEGTA was added and 20 mM KCl was replaced with 20 mM KF to eliminatecalcium to test for calcium sensitivity of channel gating. Drugs wereapplied to the intracellular side of the channel by bath perfusion.

Human non-pigmented ciliary epithelial cells were grown in tissueculture as described (Martin-Vasallo, P., Ghosh, S., and Coca-Prados,M., 1989. J. Cell. Physiol. 141, 243-252), and plated onto glass coverslips prior to use. High resistance seals (>1 Gohm) were formed betweenthe pipette and cell surface, and inside out patches were excised.Maxi-K channels in the patch were identified by their gating properties;channel open probability increased in response to membranedepolarization and elevated intracellular calcium. In patches used forpharmacological analysis, removing intracellular calcium eliminatedvoltage-gated currents. Maxi-K currents were measured after depolarizingvoltage steps or ramps that caused channel opening.

The compounds of this invention were applied to the intracellular sideof the channel in appropriate concentrations (0.001 to 100 μM). Thecompounds reduced channel open probability, and this effect was reversedupon washout of compounds from the experimental chamber. The IC50 forblock of maxi-K channels under these conditions for the compounds ofthis invention ranged from about 0.5 nM to about 10 μM.

1. A method for treating ocular hypertension or glaucoma comprisingadministration to a patient in need of such treatment a therapeuticallyeffective amount of a compound of structural formula I:

or a pharmaceutically acceptable salt, enantiomer, diastereomer ormixture thereof: wherein, R represents hydrogen, or C₁₋₆ alkyl; Xrepresents —(CHR₇)_(p)—, —(CHR₇)_(p)CO—; Y represents —CO(CH₂)_(n)—,CH₂, or —CH(OR)—; Q represents CRy; Ry represents H, or C₁₋₆ alkyl;R_(w) represents H, C₁₋₆ alkyl, —C(O)C₁₋₆ alkyl, —C(O)OC₁₋₆ alkyl,—SO₂N(R)₂, —SO₂C₁₋₆ alkyl, —SO₂C₆₋₁₀ aryl, NO₂, CN or —C(O)N(R)₂; R₂represents hydrogen, C₁₋₁₀ alkyl, OH, C₂₋₆ alkenyl, C₁₋₆ alkylSR,—(CH₂)_(n)—O(CH₂)_(m)OR, —(CH₂)_(n)C₁₋₆ alkoxy, —(CH₂)_(n)C₃₋₈cycloalkyl, —(CH₂)_(n)C₃₋₁₀ heterocyclyl, —N(R)₂, —COOR, or—(CH₂)_(n)C₆₋₁₀ aryl, said alkyl, heterocyclyl, or aryl optionallysubstituted with 1-3 groups selected from R^(a); R₃ represents hydrogen,C₁₋₁₀ alkyl, —(CH₂)_(n)C₃₋₈ cycloalkyl, —(CH₂)_(n)C₃₋₁₀ heterocyclyl,—(CH₂)_(n)COOR, —(CH₂)_(n)C₆₋₁₀ aryl, —(CH₂)_(n)NHR₈, —(CH₂)_(n)N(R)₂,—(CH₂)_(n)N(R₈)₂, —(CH₂)_(n)NHCOOR, —(CH₂)_(n)N(R₈)CO₂R,—(CH₂)_(n)N(R₈)COR, —(CH₂)_(n)NHCOR, —(CH₂)_(n)CONH(R₈), aryl,—(CH₂)_(n)C₁₋₆ alkoxy, CF₃, —(CH₂)_(n)SO₂R, —(CH₂)_(n)SO₂N(R)₂,—(CH₂)_(n)CON(R)₂, —(CH₂)_(n)CONHC(R)₃, —(CH₂)_(n)CONHC(R)₂CO₂R,—(CH₂)_(n)COR₈, nitro, cyano or halogen, said alkyl, alkoxy,heterocyclyl, or aryl optionally substituted with 1-3 groups of R^(a);or R₂ and R₃ taken together with the intervening Q form a 3-10 memberedcarbocyclic or heterocyclic carbon ring optionally interrupted by 1-2atoms of O, S, C(O) or NR, and optionally having 1-4 double bonds, andoptionally substituted by 1-3 groups selected from R^(a); or R₂ and R₃taken together with the intervening Q represent OR; R₄ and R₅independently represent hydrogen, C₁₋₆ alkoxy, OH, C₁₋₆ alkyl, COOR,SO_(q)C₁₋₆ alkyl, COC₁₋₆ alkyl, SO₃H, —O(CH₂)_(n)N(R)₂, —O(CH₂)_(n)CO₂R,—OPO(OH)₂, CF₃, OCF₃—N(R)₂, nitro, cyano, C₁₋₆ alkylamino, or halogen;and R₆ represents hydrogen, C₁₋₁₀ alkyl, —(CH₂)_(n)C₆₋₁₀ aryl,NR_(c)R_(d), —NR(CH₂)_(n)C₆₋₁₀ aryl, —N((CH₂)_(n)C₆₋₁₀ aryl),—(CH₂)_(n)C₃₋₁₀ heterocyclyl, —NR(CH₂)_(n)C₃₋₁₀ heterocyclyl,—N((CH₂)_(n)C₃₋₁₀ heterocyclyl)₂ (C₆₋₁₀ aryl)O—, —(CH₂)_(n)C₃₋₈cycloalkyl, —COOR, —C(O)CO₂R, said aryl, heterocyclyl and alkyloptionally substituted with 1-3 groups selected from R^(a), wherein theR^(a)(s) can be attached to any carbon atom or heteroatom selected fromN and 5; R_(c) and R_(d) independently represent H, C₁₋₆ alkyl, C₂₋₆alkenyl, C₁₋₆ alkylSR, —(CH₂)_(n)O(CH₂)_(m)OR, —(CH₂)_(n)C₁₋₆ alkoxy, or—(CH₂)_(n)C₃₋₈ cycloalkyl; or R_(c) and R_(d) taken together with theintervening N atom form a 4-10 membered heterocyclic carbon ringoptionally interrupted by 1-2 atoms of O, S, C(O) or NR, and optionallyhaving 1-4 double bonds, and optionally substituted by 1-3 groupsselected from R^(a); R₇ represents hydrogen, C₁₋₆ alkyl, —(CH₂)_(n)COORor —(CH₂)_(n)N(R)₂, R₈ represents —(CH₂)_(n)C₃₋₈ cycloalkyl,—(CH₂)_(n 3-10) heterocyclyl, C₁₋₆ alkoxy or —(CH₂)_(n)C₅₋₁₀ heteroaryl,—(CH₂)_(n)C₆₋₁₀ aryl said heterocyclyl, aryl or heteroaryl optionallysubstituted with 1-3 groups selected from R^(a); R^(a) represents F, Cl,Br, I, CF₃, N(R)₂, NO₂, CN, —O—, —COR₈, —CONHR₈, —CON(R₈)₂,—O(CH₂)_(n)COOR, —NH(CH₂)_(n)OR, —COOR, OCF₃, CF₂CH₂OR, —NHCOR, —SO₂R,—SO₂NR₂, —SR, (C₁-C₆ alkyl)O—, —(CH₂)_(n)O(CH₂)_(m)OR, —(CH₂)_(n)C₁₋₆alkoxy, (aryl)O—, —(CH₂)_(n)OH, (C₁-C₆ alkyl)S(O)_(m)—, H₂N—C(NH)—,(C₁-C₆ alkyl)C(O)—, (C₁-C₆ alkyl)OC(O)NH—, —(C₁-C₆alkyl)NR_(w)(CH₂)_(n)C₃₋₁₀ heterocyclyl-R_(w), —(C₁-C₆alkyl)O(CH₂)_(n)C₃₋₁₀ heterocyclyl-R_(w), —(C₁-C₆ alkyl)S(CH₂)_(n)C₃₋₁₀heterocyclyl-R_(w), —(C₁-C₆ alkyl)-C₃₋₁₀ heterocyclyl-R_(w),—(CH₂)_(n)—Z¹—C(═Z²)N(R)₂, —(C₂₋₆ alkenyl)NR_(w)(CH₂)_(n)C₃₋₁₀heterocyclyl-R_(w), —(C₂₋₆ alkenyl)O(CH₂)_(n)C₃₋₁₀ heterocycyl-R, —(C₂₋₆alkenyl)S(CH₂)_(n)C₃₋₁₀ heterocyclyl-R_(w), —(C₂₋₆ alkenyl)-C₃₋₁₀heterocyclyl-R_(w), —(C₂₋₆ alkenyl)-Z¹—C(═Z²)N(R)₂, —(CH₂)_(n)SO₂R,—(CH₂)_(n)SO₃H, —(CH₂)_(n)PO(OR)₂, C₃₋₁₀cycloalkyl, C₆₋₁₀ aryl, C₃₋₁₀heterocyclyl, C₂₋₆ alkenyl, and C₁-C₁₀ alkyl, said alkyl, alkenyl,alkoxy, heterocyclyl and aryl optionally substituted with 1-3 groupsselected from C₁-C₆ alkyl, halogen, (CH₂)_(n)OH, CN, NO₂, CON(R)₂ andCOOR; Z¹ and Z² independently represents NR_(w), O, CH₂, or S; m is 0-3;n is 0-3; p is 0-3 and q is 0-2.
 2. A method for treating macular edema,macular degeneration, increasing retinal and optic nerve head bloodvelocity, increasing retinal and optic nerve oxygen tension, and/or aneuroprotective effect comprising administration to a patient in need ofsuch treatment a pharmaceutically effective amount of a compound ofstructural formula I:

or a pharmaceutically acceptable salt, enantiomer, diastereomer ormixture thereof: wherein, R represents hydrogen, or C₁₋₆ alkyl; Xrepresents —(CHR₇)_(p)—, —(CHR₇)_(p)CO; Y represents —CO(CH₂)_(n)—, CH₂,or —CH(OR)—; Q represents CR^(y); R^(y) represents H, or C₁₋₆ alkyl;R_(w) represents H, C₁₋₆ alkyl, —C(O)C₁₋₆ alkyl, —C(O)OC₁₋₆ alkyl,—SO₂N(R)₂, —SO₂C₁₋₆ alkyl, —SO₂C₆₋₁₀ aryl, NO₂, CN or —C(O)N(R)₂; R₂represents hydrogen, C₁₋₁₀ alkyl, OH, C₂₋₆ alkenyl, C₁₋₆ alkylSR,—(CH₂)_(n)—O(CH₂)_(m)OR, —(CH₂)_(n)C₁₋₆ alkoxy, —(CH₂)_(n)C₃₋₈cycloalkyl, —(CH₂)_(n)C₃₋₁₀ heterocyclyl, —N(R)₂, —COOR, or—(CH₂)_(n)C₆₋₁₀ aryl, said alkyl, heterocyclyl, or aryl optionallysubstituted with 1-3 groups selected from R^(a); R₃ represents hydrogen,C₁₋₁₀ alkyl, —(CH₂)_(n)C₃₋₈ cycloalkyl, —(CH₂)_(n)C₃₋₄₀ heterocyclyl,—(CH₂)_(n)COOR, —(CH₂)_(n)C₆₋₁₀ aryl, —(CH₂)_(n)NHR₈, —(CH₂)_(n)N(R)₂,—(CH₂)_(n)N(R₈)₂, —(CH₂)_(n)NHCOOR, —(CH₂)_(n)N(R₈)CO₂R,—(CH₂)_(n)N(R₈)COR, —(CH₂)_(n)NHCOR, —(CH₂)_(n)CONH(R₈), aryl,—(CH₂)_(n)C₁₋₆ alkoxy, CF₃, —(CH₂)_(n)SO₂R, —(CH₂)_(n)SO₂N(R)₂,—(CH₂)_(n)CON(R)₂, —(CH₂)_(n)CONHC(R)₃, —(CH₂)_(n)CONHC(R)₂CO₂R,—(CH₂)_(n)COR₈, nitro, cyano or halogen, said alkyl, alkoxy,heterocyclyl, or aryl optionally substituted with 1-3 groups of R^(a);or R₂ and R₃ taken together with the intervening Q form a 3-10 memberedcarbocyclic or heterocyclic carbon ring optionally interrupted by 1-2atoms of O, S, C(O) or NR, and optionally having 1-4 double bonds, andoptionally substituted by 1-3 groups selected from R^(a); or R₂ and R₃taken together with the intervening Q represent OR; R₄ and R₅independently represent hydrogen, C₁₋₆ alkoxy, OH, C₁₋₆ alkyl, COOR,SO_(q)C₁₋₆ alkyl, COC₁₋₆ alkyl, SO₃H, —O(CH₂)_(n)N(R)₂, —O(CH₂)_(n)CO₂R,—OPO(OH)₂, CF₃, OCF₃—N(R)₂, nitro, cyano, C₁₋₆ alkylamino, or halogen;and R₆ represents hydrogen, C₁₋₁₀ alkyl, —(CH₂)_(n)C₆₋₁₀ aryl,NR_(c)R_(d), —NR(CH₂)_(n)C₆₋₁₀ aryl, —N((CH₂)_(n)C₆₋₁₀ aryl)₂,—(CH₂)_(n)C₃₋₁₀ heterocyclyl, —NR(CH₂)_(n)C₃₋₁₀ heterocyclyl,—N((CH₂)_(n)C₃₋₁₀ heterocyclyl)₂(C₆₋₁₀ aryl)O—, —(CH₂)_(n)C₃₋₈cycloalkyl, —COOR, —C(O)CO₂R, said aryl, heterocyclyl and alkyloptionally substituted with 1-3 groups selected from R^(a), wherein theR^(a)(s) can be attached to any carbon atom or heteroatom selected fromN and S; R_(c) and R_(d) independently represent H, C1-6 alkyl, C₂₋₆alkenyl, C₁₋₆ alkylSR, —(CH₂)_(n)O(CH₂)_(m)OR, —(CH₂)_(n)C₁₋₆ alkoxy, or—(CH₂)_(n)C₃₋₈ cycloalkyl; or R_(c) and R_(d) taken together with theintervening N atom form a 4-10 membered heterocyclic carbon ringoptionally interrupted by 1-2 atoms of O, S, C(O) or NR, and optionallyhaving 1-4 double bonds, and optionally substituted by 1-3 groupsselected from R^(a); R₇ represents hydrogen, C₁₋₆ alkyl, —(CH₂)_(n)COORor —(CH₂)_(n)N(R)₂, R₈ represents —(CH₂)_(n)C₃₋₈ cycloalkyl,—(CH₂)_(n 3-10) heterocyclyl, C₁₋₆ alkoxy or —(CH₂)_(n)C₅₁o heteroaryl,—(CH₂)_(n)C₆₋₁₀ aryl said heterocyclyl, aryl or heteroaryl optionallysubstituted with 1-3 groups selected from R^(a); R^(a) represents F, Cl,Br, I, CF₃, N(R)₂, NO₂, CN, —O—, —COR₈, —CONHR₈, —CON(R₈)₂,—O(CH₂)_(n)COOR, —NH(CH₂)_(n)OR, COOR, —OCF₃, CF₂CH₂OR, NHCOR, SO₂R,SO₂NR₂, SR, (C₁-C₆ alkyl)O—, —(CH₂)_(n)—O(CH₂)_(m)OR, —(CH₂)_(n)C₁₋₆alkoxy, (aryl)O—, —(CH₂)_(n)OH, (C₁-C₆ alkyl)S(O)_(m)—, H₂N—C(NH)—,(C₁-C₆ alkyl)C(O)—, (C₁-C₆ alkyl)OC(O)NH—, —(C₁-C₆alkyl)NR_(w)(CH₂)_(n)C₃₋₁₀ heterocyclyl-R_(w), —(C₁-C₆alkyl)O(CH₂)_(n)C₃₋₁₀ heterocyclyl-R_(w), —(C₁-C₆ alkyl)S(CH₂)_(n)C₃₋₁₀heterocyclyl-R_(w), —(C₁-C₆ alkyl)-C₃₋₁₀ heterocyclyl-R_(w),—(CH₂)_(n)—Z¹C(═Z²)N(R)₂, —(C₂₋₆ alkenyl)NR_(w)(CH₂)_(n)C₃₋₁₀heterocyclyl-R_(w), —(C₂₋₆ alkenyl)O(CH₂)_(n)C₃₋₁₀ heterocyclyl-R_(w),—(C₂₋₆ alkenyl)S(CH₂)_(n)C₃₋₁₀ heterocyclyl-R_(w), —(C₂₋₆ alkenyl)-C₃₋₁₀heterocyclyl-R_(w), —(C₂₋₆ alkenyl)-Z¹—C(═Z²)N(R)₂, —(CH₂)_(n)SO₂R,—(CH₂)_(n)SO₃H, —(CH₂)_(n)PO(OR)₂, C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, C₃₋₁₀heterocyclyl, C₂₋₆ alkenyl, and C₁-C₁₀ alkyl, said alkyl, alkenyl,alkoxy, heterocyclyl and aryl optionally substituted with 1-3 groupsselected from C₁-C₆ alkyl, halogen, (CH₂)_(n)OH, CN, NO₂, CON(R)₂ andCOOR; Z¹ and Z² independently represents NR, O, CH₂, or S; m is 0-3; nis 0-3; p is 0-3 and q is 0-2.
 3. The method in accordance with claim 1wherein the compound is represented by:

wherein: R1 R2

or a pharmaceutically acceptable salt, enantiomer, diastereomer ormixture thereof.
 4. The method according to claim 3 wherein the compoundis

or a pharmaceutically acceptable salt, enantiomer, diastereomer ormixture thereof.